P. carinii infections remain common in HIV-infected patients despite the broad use of highly active antiretroviral therapies and prophylactic regimens. Studies of human P. carinii are focusing on two areas: diagnosis and evaluation for potential resistance to therapy. To try to develop highly sensitive non-invasive diagnostic methods, we have been evaluating polymerase chain reaction (PCR) using primers based on the major surface glycoprotein (MSG) genes of human P. carinii. This is a family of genes that are closely related and that encode an important surface protein of P. carinii. PCR using primers based on this gene is potentially a highly sensitive method since this is a multicopy gene (estimated at >100 copies/genome). We have been evaluating the diagnostic potential using a conserved region of the gene family. Our studies have shown that the sensitivity of MSG-based primers is greater than that of previously utilized primers. We are currently evaluating these primers prospectively in collaboration with the Microbiology Department. With regard to resistance, because human P. carinii cannot be cultured, we cannot directly determine if resistance to commonly used therapeutic agents is developing. However, molecular techniques can be used to identify mutations that may confer resistance in genes that are targets of therapeutic agents. The most commonly used agent to treat P. carinii pneumonia is the combination of trimethoprim, which targets dihydrofolate reductase (DHFR), and sulfamethoxazole, which targets dihydropteroate synthase (DHPS). We and others have recently identified mutations in the active site of the human P. carinii DHPS gene in patients who have received TMP SMX for prophylaxis. We have cloned the human P. carinii DHFR gene, and have exam-ined (by PCR and sequencing) the P. carinii DHFR and DHPS genes of a variety of human isolates from patients with P. carinii pneumonia. DHPS mutations were found in about one-third of patients, while no mutations have been found to date in the DHFR gene. These studies should provide improved diagnostic methods for PCP as well as insights into the reasons for therapy or prophylaxis failures.